HIV-2 transmembrane glycoprotein homodimer (gp 80)

ABSTRACT

Characterization of the envelope transmembrane protein of human immunodeficiency virus type 2 (HIV-2) was carried out using murine polyclonal and monoclonal antibodies or patient sera specific for HIV-2 proteins. A 80-Mr glycoprotein (gp80) was produced in HIV-2 infected cells along with three other glycoproteins that were recently reported: the extracellular glycoprotein (gp125), the envelope glycoprotein precursor (gp140), and the transient dimeric form of gp140 (gp300). The gp125 and gp80 were detectable after the synthesis of gp140 and the formation of gp300. Among these four glycoproteins, only gp80 and gp125 were associated with HIV-2 virions. As the other glycoproteins, gp80 was recognized by all HIV-2 positive sera. A murine polyclonal antibody raised against the purified gp300 recognized all four glycoproteins. On the other hand, a monoclonal antibody raised against a synthetic polypeptide deduced from the sequence of the transmembrane glycoprotein of HIV-2, recognized gp140, gp300 and gp80; thus indicating that gp80 should be related to the transmembrane protein of the envelope. Dimerization of envelope glycoprotein precursor and the transmembrane glycoprotein was also observed in cells infected with simian immunodeficiency virus (SIV), a virus closely related to HIV-2. Dimerization of the envelope precursors might be essential for the processing of these glycoproteins into the mature products extracellular and transmembrane glycoproteins. Furthermore the dimeric form of the transmembrane glycoproteins might be important for the optimal structure of the virus and thus for its infectivity.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part of application SerialNo. 204,346, filed Jun. 9, 1988, (Attorney Docket No. PAST-068), theentire disclosure of which is relied upon and incorporated by referenceherein.

BACKGROUND OF THE INVENTION

[0002] This invention relates to viral proteins and glycoproteins, tocompositions containing these proteins, to methods of preparing theproteins, and to their use in detecting viral infection.

[0003] Human immunodeficiency virus (HIV) is the etiological agent ofacquired immunodeficiency syndrone (AIDS) (Montagnier et al., 1984). Todate, two related but distinct viruses HIV-1 and HIV-2, have beenidentified (Barre-Sinoussi et al., 1983; Brun-Vezinet et al., 1987;Clavel et al., 1986a, 1986b; Guyader et al., 1987; Popovic et al., 1984;Ratner et al., 1985; Wain-Hobson et al., 1985). HIV-2 is closely relatedto simian immunodeficiency virus (SIV-mac), which causes an AIDS-likedisease in macaques (Daniel et al., 1985; Fultz et al., 1986;Chakrabarti et al., 1987). Alignments of the nucleotide sequences ofHIV-1, HIV-2, and SIV reveal a considerable homology between HIV-2 andSIV-mac. These two viruses share about 75% overall nucleotide sequencehomology, but both of them are only Distantly related to HIV-1 withabout 40% overall homology (Guyader et al., 1987; Chakrabarti et al.,1977).

[0004] In addition to the genes that encode structural proteins (thevirion capsid and envelope glycoproteins) and the enzymes required forproviral synthesis and integration common to all retroviruses, HIV-1,HIV-2, and SIV encode genes that regulate virus replication as well asgenes that encode proteins of yet unknown function. The only notabledifference in the genetic organizations of HIV-1, HIV-2, and SIV residesin the open reading frame referred to as vox, which is absent in HIV-1and vou in HIV-1 but not in HIV-2 and SIV (Cohen et al., 1988; Guyaderet al., 1987). These viruses are both tropic and cytopathic for CD4positive T lymphocytes (Klatzmann et al., 1984; Clavel et al., 1985a;Dalgleish et al., 1984; Daniel et al., 1985). A great number of studieshave indicated that CD4 functions as the cellular receptor of HIV(Weiss, 1988).,

[0005] The HIV-1 env gene encodes for a 160-kilodalton (kDa)glycoprotein that is proteolytically cleaved to yield the extracellularand transmembrane proteins, gp120 and gp41, respectively (Montagnier etal., 1985). Similarly, HIV-2 env gene encodes for a precursorglycoprotein which is then processed to the mature extracellular andtransmembrane glycoproteins (Rey et al., 1989). However, unlike HIV-1,the processing of HIV-2 envelope precursor gp140 seems to require theformation of a homologous dimer (gp300) during its proceessing.Interestingly, dimerization of the envelope precursor is also observedin SIV infected cells (Rey et al. 1989). Accordingly, dimer formationseems to be a specific property of HIV-2 and SIV envelope geneexpression.

[0006] There exists a need in the art for additional information on thestructure and in vivo processing of HIV-2 proteins, and especially HIV-2envelope proteins and glycoproteins. Such information would aid inidentifying HIV-2 infection in individuals. In addition, such findingscould aid in elucidating the mechanism by which HIV-2 infection andvirus proliferation occur and thereby make it possible to devise modesof intervening in viral processes.

SUMMARY OF THE INVENTION

[0007] This invention aids in fulfilling these needs in the art byproviding HIV-2 envelope proteins and glycoproteins in purified form.More particularly, this invention relates to the processing of HIV-2envelope glycoproteins and the characterization of the transmembraneglycoprotein. Previously, the detection of the transmembraneglycoprotein had been handicapped by the lack of specific antibodies.For this reason, polyclonal antibodies were prepared against thepurified HIV-2 envelope precursor. Furthermore, monoclonal antibodieswere prepared against a synthetic polypeptide deduced from the sequenceof the transmembrane glycoprotein of HIV-2. With the help of theseantibodies the membrane glycoproteins of HIV-2 and SIV were identified.

[0008] It was discovered that the transmembrane proteins exist as ahomodimer in the infected cells as well as in the virions. Dimeric formsof the transmembrane glycoproteins of HIV-2 and SIV can be dissociatedin an ionic detergent to 36 kDa and 32 kDa proteins, respectively.Conformational modifications brought about by the formation of envelopeprecursor might be necessary for transport of the glycoprotein precursorto the Golgi apparatus and its processing into the mature glycoproteinproducts, ate extracellular and transmembrane envelope proteins.Futhermore, the transmembrane dimer might be essential for optimalstructure of the virion and thus its infectivity.

[0009] This invention thus provides gp80 structural glycoprotein ofHIV-2 dimeric form of the transmembrane glycoprotein and humanretroviral variants of HIV-2 containing the structural glycoprotein inpurified form.

[0010] A similar high molecular weight glycoprotein of Simianimmunodeficiency Virus (SIV) or of a Simian retroviral variant of SIVhas also been discovered. This glycoprotein is the dimeric form oftransmembrane glycoprotein of SIV and has an apparent molecular weightof about 65 kDa (gp65SIV). This glycoprotein is also provided in apurified form.

[0011] This invention also provides labeled gp80 of HIV-2 and gp65 ofSIV. Preferably, the labeled glycoproteins are in purified form. It isalso preferred that the labeled glycoprotein is capable of beingimmunologically recognized by human body fluid containing antibodies toHIV-2 or SIV. The glycoproteins can be labeled, for example, with animmunoassay label selected from the group consisting of radioactive,enzymatic, fluorescent, chemiluminescent labels, and chromophores.

[0012] Immunological complexes between the proteins and glycoproteins ofthe invention and antibodies recognizing die proteins and glycoproteinsare also provided. The immunological complexes can be labeled with animmunoassay label selected from the group consisting of radioactive,enzymatic, fluorescent, chemiluminescent labels, and chromophores.

[0013] Furthermore, this invention provides a method for detectinginfection of cells by human immunodeficiency virus type-2 (HIV-2). Themethod comprises providing a composition comprising cells suspected ofbeing infected with HIV-2, disrupting cells in the composition to exposeintracellular proteins, and assaying the exposed intracellular proteinsfor the presence of gp80 glycoprotein of HIV-2. The exposedintracellular proteins are typically assayed by electrophoresis or byimmunoassay with antibodies that are immunologically reactive with gp80glycoprotein of HIV-2.

[0014] This invention provides still another method of detectingantigens of HIV-2, which comprises providing a composition suspected ofcontaining antigens of HIV-2, and assaying the composition for thepresence of gp80 glycoprotein of HIV-2. The composition is typicallyfree of cellular debris. The molecular weight of the gp80 is estimatedmore or less 10%. The same for the other molecular weight mentioned inthe present invention.

[0015] A method of distinguishing HIV-2 infection from HIV-1infection-in-cells suspected of being infected therewith has also beendiscovered. The method comprises providing an extract containingintracellular proteins of the cells, and assaying the extract for thepresence of gp80 glycoprotein. The gp80 is characteristic of HIV-2, butthe glycoprotein has not been found in extracts of HIV-1 cell cultures.

[0016] In addition, this invention provides a method of making gp80glycoprotein of HIV-2, which comprises providing a compositioncontaining cells in which HIV-2 is capable of replicating, infecting thecells with HIV-2, and culturing the cells under conditions to causeHIV-2 to proliferate. The cells are then disrupted to exposeintracellular proteins. The gp80 glycoprotein is recovered from theresulting exposed intracellular proteins. It could be also recovered bydetergent solubilization of HIV-2 virions.

[0017] This invention also provides an in vitro diagnostic method forthe detection of the presence or absence of antibodies which bind to anantigen comprising the proteins or glycoproteins of the invention ormixtures of the proteins and glycoproteins. The method comprisescontacting the antigen with a biological fluid for a time and underconditions sufficent for the antigen and antibodies in the biologicalfluid to form an antigen-antibody complex, and then detecting theformation of the complex. The detecting step can further comprisemeasuring the formation of the antigen-antibody complex. The formationof the antigen-antibody complex is preferably measured by immunoassaybased on Western Blot technique, ELISA (enzyme linked immunosorbentassay), indirect immunofluourescent assay, or immunoprecipitation assay.

[0018] A diagnostic kit for the detection of the presence or absence ofantibodies which bind to the proteins or glycoproteins of the inventionor mixtures of the proteins and glycoproteins contains antigencomprising the proteins, glycoproteins, or mixtures thereof and meansfor detecting the formation of immune complex between the antigen andantibodies. The antigens and the means are present in an amountsufficient to perform the detection.

[0019] This invention provides a method of preparing envelopetransmembrane glycoproteins, which comprises providing an extracellularcomposition containing gp80 glycoprotein of HIV-2 or gp65 of SIV andthen dissociating the glycoprotein of HIV-2 or the glycoprotein of SIV.A non-glycosylated dimeric form of the transmembrane envelope protein ofHIV-2 (and SIV) can be obtained from the glycosylated form of gp80 (orgp65) by using specific enzymes (i.e. endo F), which cleave matureoligosaccharide chains. Another procedure for the production ofunglycosylated forms of such dimeric protein could be geneticengineering methods (see Reference 16).

[0020] This invention also provides an immunogenic compositioncomprising a protein or glycoprotein of the invention in an amountsufficient to induce an immunogenic or protective response an vivo, inassociation with a pharmaceutically acceptable carrier therefor. Avaccine composition of the invention comprises a neutralizing amount ofthe dimeric transmembrane envelope glycoprotein or unglycosylated formthereof and a pharmaceutically acceptable carrier therefor.

[0021] The dimeric form of the transmembrane glycoprotein is highlyrecognized by all sera positive for HIV-2 antigens. Therefore, thedetection of gp80 could be used as a marker for characterization ofHIV-2 positive sera and for differentiation from HIV-1 positive sera.

[0022] The proteins and glycoprotein of this invention are thus usefulas a portion of a diagnostic composition for detecting the presence ofantibodies to antigenic proteins associated with HIV-2 and SIV. Inaddition, the proteins and glycoproteins can be used to raise antibodiesfor detecting the presence of antigenic proteins associated with HIV-2and SIV. The proteins and glycoproteins of the invention can be alsoemployed to raise neutralizing antibodies that either inactivate thevirus, reduce the viability of the virus in vivo, or inhibit or preventviral replication. The ability to elicit virus-neutralizing antibodiesis especially important when the proteins and glycoproteins of theinvention are used in vaccinating compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] This invention will be described in greater detail by referringto the drawings in which:

[0024]FIG. 1 is an autoradiograph of a specific 80-kDa protein in HIV-2infected cells western blot analysis was made using an HIV-1 positiveserum and 3 HIV-2 positive sera (A, B and C). Extracts (material from10⁶ cells) from control uninfected (lanes 2), HIV-1 infected (lanes 1),and HIV-2 infected (lanes 3) CEM cells were analyzed by polyacrylamidegel (7%) electrophoresis before the western blot assay. On the left, thearrows indicate the position of HIV-1 extracellular glycoprotein (gp120)and aq precursors p55 and p40. On the right is the position of HIV-2specific gp300, gp140, and gp80.

[0025]FIG. 2 is a fluorograph relating to synthesis of HIV-2 relatedglycoproteins. HIV-2 infected cells were labeled with [³H] glucosamine(200 μCi/ml; 4×10⁶ cells/ml) for 2, 3, 4, 6, and 8 hr. At differentpoints, extracts (material from 10⁷ cells) were prepared from infectedcells and from the virus pellet (prepared by 100,000 g centrifugation ofthe culture medium). Aliquots from these extracts (corresponding to2×10⁶ cells) were purified on HIV-2 serum-Sepharose, and the labeledproteins were analyzed by polyacrylamide gel (12.5%) electrophoresis. Onthe left is the position of protein mol. wt. markers: myosin, 200,000;phosphorylase B, 97,000; bovine serum albumin, 68,000; ovalbumin,43,000; carbonic anhydrase, 30,000.

[0026]FIG. 3 depicts a Western blot analysis with polyclonal antibodiesagainst gp300. On the left, extracts from uninfected (−) and HIV-1 orHIV-2 infected CEM cells are shown. On the right, extracts from HIV-2infected CEM cells are shown: cell extracts (lane C) and virus pellet(lane V). These samples were analyzed by polyacrylamide gelelectrophoresis (7.5% gel on the left; 12.5% gel on the right) beforewestern blot analysis using murine polyclonal antibodies raised againstthe purified gp300 (anti-gp300). An autoradiogram is shown. Each samplecorresponded to material from 10⁶ cells.

[0027]FIG. 4 depicts a Western blot analysis using the monoclonalantibody mAb 1H8. Extracts from HIV-1 or HIV-2 infected cells (lanes C)and virus pellet (lanes V) were analyzed by polyacrylamide gel (12.5%)electrophoresis before Western blot assay using mAb 1H8. Anautoradiograph is shown. This monoclonal antibody was raised againstsynthetic peptide p39′ of the purified HIV-2 virus and it is directedagainst the transmembrane glycoprotein of HIV-2 envelope.

[0028]FIG. 5 shows that peptide p39′ blocks the binding of mAb 1H8 togp80. Extracts from the HIV-2 virus pellets were analyzed by Westernblot assay using mAb 1H8 (section mAb) or anti-gp300 polyclonalantibodies (section S). Incubation with each antibody was carried out inthe absence (lanes −) or presence (lanes +) of 10 μg of peptide p39′.The results of the autoradiography are shown.

[0029]FIG. 6 depicts the results of pulse chase experiments to show theproduction of gp80 in HIV-2 infected cells. HIV-2 infected CEM cellswere labeled with [³⁵] methionine (200 μCi/ml; 4×10⁶ cells/ml) for 2 hr(lanes 0). The radioactive label was then chased in culture mediumcontaining 5 mM cold methionine for 2 and 4 hr (lanes 2 and 4). Theculture medium at 4 hr was centrifuged at 100,000 g and the pellet wasextracted. All samples were immunoprecipitated using anti-gp300 or mAb1H8 antibodies labeled proteins in the immune complex preparations wereeluted in the electrophoresis sample Duffer and analyzed bypolyacrylamide gel (7.5%) electrophoresis. A fluorograph is shown. C andV stand for cell and virus extracts, respectively. Each samplerepresents material from 10⁶ cells.

[0030]FIG. 7 shows (a) incorporation of labeled glucosamine and fucoseinto gp80; and (b) the effect of castanospermine on the production ofgp125 and gp80. (a) HIV-2 infected CEM cells labeled with [³H]glucosamine (200 μCi/ml or with [³H] fucose (200 μCi/ml) were assayed byiimmunoprecipitation using anti-gp300 polyclonal antibodies (lanes S) orthe monoclonal antibody mAb 1H8 (lanes M). All samples were analyzed bypolyacrylamide gel (12.5%) electrophoresis. A fluorograph is shown. (b)HIV-2 infected cells were labeled (16 hr) with [35S] methionine (200μCi/ml; 4×10⁶ cells/ml) in the absence (lane −) or presence (lanes +) ofcastanosphermine (1 mM). Extracts from the culture medium containingvirus particles were purified by immunoaffinity column using HIV-2serum-Sepharose, and the purified proteins were assayed bypolyacrylamide gel (12.5%) electrophoresis. A fluorograph is shown.

[0031]FIG. 8 relates to dissociation of gp80. Section C; extracts from[35S] methionine labeled, HIV-2 infected CEM cells were assayed byimmunoprecipitation using the monoclonal antibody mAb 1H8 (lane 1).Another aliquot of the same cell extract preparation was first heated(95° C., 5 min) in the presence of 1% SDS, then it was diluted 10 foldin RIPA buffer before the immunoprecipitation assay (lane 2). The immunecomplex preparations were analyzed by electrophoresis. Each sample wasfrom extracts corresponding 10⁶ cells. Section V: HIV-2 virus pelletsfrom [35S] methionine labeled cells (each corresponding to material from10⁷ cells) were suspended in different buffers: (1) lysis buffercontaining Triton (10 mM Tris-HCl pH 7.6, 150 mM NaCl, 1 mm EDTA, 1%(v/v) Triton X-100 and 100 units/ml aprotinin); (2) lysis buffercontaining SDS (as in 1, but containing 1% (v/v) SDS instead TritonX-100); (3) lysis buffer containing SDS and then heated (95° C., 5 min);(4) RIPA buffer (as in 1, but also containing 0.1% (v/v) SDS and 0.2%(v/v) deoxycholate). All these samples were then immunoprecipitatedusing mAb 1H8 and labeled proteins were analyzed by polyacrylamide gel(12.5%) electrophoresis. A fluorograph is shown.

[0032]FIG. 9 relates to dissociation of the purified gp80 into gp36.HIV-2 infected CEM cells were labeled (17 hr) with [35S] methionine andthe virus pellets were suspended is lysis buffer containing Triton.These virus extracts (material corresponding from 2×10⁷ cells) wereimmunoprecipitated using mAb 1H8, and gp80 was purified by preparativegel electrophoresis. Equal aliquots of the purified gp80 preparationwere lyophilized and suspended in 100 mM acetate at pH 6.8, 5.8, and 4.8containing 1% (v/v) SDS, 100 units/ml aprotinin and 5 mM EGTA (toinhibit calcium-dependent proteolysis). All the samples were incubatedat 30° C. for 50 min before dilution in 2 fold concentratedelectrophoresis buffer. Samples were analyzed by polyacrylamide gel(12.5%) electrophoresis. A fluorograph is shown.

[0033]FIG. 10 substantiates that the transmembrane glycoprotein of SIVexists as a dimer. Section Cell: SIV-mac infected HUT-3 cells and HIV-2infected CEM cells were labeled for 16 hr with [³H] glucosamine (200μCi/ml; 4×10⁶ cells/ml). Extracts (prepared in lysis buffer containingTriton) from infected cells Were purified by immunoprecipitation usingmAb 1H8 and the labeled proteins were analyzed by polyacrylamide gel(12.5%) electrophoresis. A fluorograph is shown.

[0034]FIG. 11 is a schematic pathway of HIV-2 envelope glycoproteinprocessing.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0035] As a result of this invention, the processing of HIV-2 envelopeglycoproteins has now been characterized. An 80-Mr glycoprotein (gp80)was produced in HIV-2 infected cells along with three otherglycoproteins that were recently reported: the extracellularglycoprotein (gp125), the envelope glycoprotein precursor (gp140), andthe transient dimeric form of gp140 (gp300).

[0036] The gp125 and gp80 were detectable after the synthesis of gp140and the formation of gp300. Among these four glycoproteins, only gp80and gp125 were associated with HIV-2 virions. As the otherglycoproteins, gp80 was recognized by all HIV-2 positive sera. A murinepolyclonal antibody raised against purified gp300 recognized all fourglycoproteins. On the other hand, a monoclonal antibody specific fortransmembrane glycoprotein of HIV-2 recognized gp140, gp300, and gp80,thus indicating that gp80 should be related to the transmembrane proteinof the envelope. Heating (95° C., 5 min) of cellular or viral extractsin 1% SDS resulted in the dissociation of gp80 into the monomer gp36.These results suggest that during the processing of the HIV-2 envelopeglycoprotein, the dimeric form of the precursor becomes cleaved by thecellular protease to give the extracellular glycoprotein gp125 and thetransmembrane glycoprotein dimer gp80.

[0037] Dimerization of envelope glycoprotein precursor and thetransmembrane glycoprotein was also observed in cells infected withsimian immunodeficiency virus (SIV), a virus closely related to HIV-2.Dimerization of the envelope precursor might be required for itsprocessing to give the mature envelope proteins, whereas thetransmembrane dimer might be essential for optimal structure of thevirion.

[0038] The results obtained in practicing this invention will now bedescribed in greater detail.

[0039] I. Detection of a 80 kDa Protein in HIV-2 Infected Cells and inthe Virion

[0040] Recently, we reported that the precursor of HIV-2 envelopeglycoproteins is a 140-kDa protein (gp140), which requires the formationof a homologous dimer during its processing into the nature products,the extracellular (gp125) and transmembrane (gp36) glycoproteins (Rey etal., 1989). In these studies, however, the level of gp36 was found to bevery low and in some experiments it was not detectable. It has now beendiscovered that, in fact, that is the case because the transmembraneglycoprotein exists as a homodimer with an electrophoretic mobility inpolyacrylamide gels at a position corresponding to a 80-kDa protein(FIGS. 1 to 6). Accordingly for convenience, this 80-kDa protein will bereferred to as gp80.

[0041] Crude extracts from uninfected or HIV-1_(BRU) and HIV-2_(ROD)infected CEM cells were analyzed by an electrophoretic transferimmunoblotting assay (Western blot) using an HIV-1 positive serum andthree different HIV-2 positive sera from AIDS patients (FIG. 1). TheHIV-2 specific sera identified the envelope precursors (gp140 and gp300)and in addition recognized strongly the 80-kDa protein (gp80). Thesesera were specific for HIV-2 proteins since they did not recognize HIV-1proteins which were detectable using HIV-1 specific serum: the envelopeglycoprotein precursor (gp160) and qaq precursors (p55 and p40). Therelation of gp80 to HIV-2 infection was demonstrated by several resultsin which gp80 was not identified by HIV-1 positive serum nor was itfound in HIV-1 infected cells.

[0042] Western blot analysis of viral pellets prepared by centrifugation(100,000 g for 30 min) of infected culture medium indicated that gp80was also detectable in HIV-2 particles along the extracellularglycoprotein, gp125 (see below).

[0043] II. Synthesis of gp80 in HIV-2 Infected Cells

[0044] Preliminary experiments indicated that all HIV-2 positive seracan immunoprecipitate gp80 in addition to the envelope precursors gp140and gp300 and the extracellular glycoprotein, gp125. In order tocharacterize the synthesis of gp80, an HIV-2 positive serum whichrecognizes mainly the envelope proteins was used. Purified antibodiesfrom this serum were coupled to CNBr activated Sepharose (HIV-2serum-Sepharose) and was used as an immunoaffinity column to purifyenvelope glycoproteins. HIV-2 infected cells were labeled with [³H]glucosamine, and at different times (2, 3, 4, 6, and 8 hr) extracts wereprepared from infected cells as well as from virus pellets. All sampleswere purified on HIV-2 serum-Sepharose and labeled proteins wereanalyzed by polyacrylamide gel electrophoresis (FIG. 2). At 2 hr, gp140and gp300 were the only labeled proteins detectable in infected cells;gp125 and gp80 became detectable 3 to 4 hr after the start of thelabeling during which time they became also detectable in virus pelletsprepared from the culture medium. At 6 to 8 hours after the start oflabeling, gp125 and gp80 became clearly detectable. These resultsindicate, therefore, that gp80 is associated with virus particles andsuggest that gp80 might be a mature product of a precursor whichrequires processing. In these experiment, we could also detect some [³H]glucosamine labeled gp36, but only intracellularly. The identity of thelabeled 200 kDa protein is not known (FIG. 2). It is probably a cellularprotein since it was not immunoprecipitated by other HIV-2 positivesera.

[0045] These kinetics results for the synthesis of HIV-2 envelopeglycoproteins are in accord with previous results. In HIV-2 infectedcells, gp140 is the first envelope product detectable at 15 min after apulse-labeling. During a period of chase, the dimeric form of theenvelope precursor (gp300) becomes detectable at 0.5 hr, whereas themature extracellular glycoprotein (gp125) becomes detectable at 1.5 to 3hr (Rey et al., 1989).

[0046] III. Identification of gp80 by Polyclonal Antibodies AgainstHIV-2 Envelope Precursor

[0047] In order to characterize HIV-2 envelope glycoproteins, polyclonalantibodies against the purified dimeric precursor, gp300, were prepared.For this purpose, gp300 was first partially purified by animmunoadsorbent with antibodies from HIV-2 seropositive patient serumbefore purification by preparative electrophoresis. Five mice wereimmunized with 5 μg of this purified gp300 preparation administeredintraperitoneally five times at 10 days interval. Poly(A).poly(U) (200μg) were used as an adjuvant which was administered mixed with theantigen (Materials and Methods), infra (see page 36). All mice developedantibodies against gp300. These antibodies are referred to as anti-gp300polyclonal antibodies.

[0048]FIG. 3 shows a Western blot analysis using antibodies from one ofthe immunized mice. Anti-gp300 antibodies reacted specifically withgp300, but also with gp140, gp125, and gp80 present in HIV-2 infectedcells. No specific signal was observed in uninfected or HIV-1 infectedCEM cells. The labeling of a 60 kDa protein with anti-gp300 antibodieswas probably not specific since it was observed in cell extractsirrespective of virus infection (FIG. 3, section Cells) and in someexperiments it was not at all observed.

[0049] These polyclonal antibodies were also used in a similar Westernblot assay using extracts from HIV-2 infected cells as well as from thevirus pellet. In the cellular extracts, the antibodies recognized gp300,gp140, and gp80 (FIG. 3, section HIV-2 lane C). In the viral extracts,they recognized gp125, gp80, and a 36 kDa protein which is probably thetransmembrane glycoprotein, gp36 (FIG. 3, section HIV-2 lane V). Onprolonged exposures, it was also possible to see a signal at theposition of gp36 in cellular extracts (data not shown). It isinteresting here to note that the level of gp80 and gp36 was much higherin the viral pellet compared to the cellular extract.

[0050] These results indicate that polyclonal antibodies raised againstthe envelope precursor identify gp80 along with all the components ofHIV-2 envelope. Thus, gp80 should be related to HIV-2 envelope. The factthat gp80 is associated with the virus suggests that it is a matureproduct.

[0051] IV. Identification of gp80 by the Monoclonal Antibody 1H8Specific for the Transmembrane Glycoprotein of HIV-2

[0052] The monoclonal antibody (mAb 1H8) was used in a Western blotassay to determine whether viral proteins can be identified. In HIV-2infected cells, mAb 1H8 identified gp300, gp140, and gp80, whereas inthe HIV-2 pellet it identified mainly gp80 and weakly gp36 and gp300(FIG. 4). The presence of low levels of gp300 in the virus pellet wasprobably due to some contamination from lysed cells, since it is acellular protein (Rey et al., 1989). The weak signal with gp36 probablyreflects low levels of this protein. The mAb 1H8 did not recognizeproteins in extracts from HIV-1 infected cells or from the virus pellet.Furthermore, it did not recognize the extracellular glycoprotein gp125(FIG. 4). These results illustrate, therefore, the specificity of mAb1H8 for HIV-2 envelope precursors (gp140 and gp300) and thetransmembrane glycoprotein (gp36). The reactivity of mAb 1H8 was mappedto the amino acid sequence 579-604 within the HIV-2 transmembraneglycoprotein using a synthetic peptide referred to as p39′.

[0053] To show the specificity of mAb 1HS reactivity with gp80, aWestern blot assay using extracts from HIV-2 virus pellet was carriedout. After the transfer of proteins, nitrocellulose sheets wereincubated with mAb 1H8 or anti-gp300 polyclonal antibodies in theabsence or presence of 1 μg/ml of peptide p39′ (FIG. 5). The mAb 1H8gave a strong signal for gp80. In addition, a signal for gp36 wasobserved, but only after a prolonged exposure of the autoradiogram.Anti-gp300 polyclonal antibodies reacted with gp125, gp80, and gp36; the60-kDa signal was not specific (as in FIG. 3). Addition of peptide p39′completely abolished the signals obtained with mAb 1H8, but not thoseobtained with anti-gp300 antibodies (FIG. 5). These observations confirmthat the reactivity of mAb 1H8 should be with the 26 amino acid residuecorresponding to the amino acids 579 to 604 in the transmembraneglycoprotein of HIV-2. Consequently, a sequence corresponding to that ofpeptide p39′ should be present in gp80. The reactivity of anti-gp300antibodies was not modified by peptide p39′. Therefore, these antibodiesshould interact with other epitopes than that corresponding to peptide39′.

[0054] V. Immunoprecipitation of gp80 by anti-gp300 PolyclonalAntibodies and by mAb 1H8

[0055] Anti-gp300 polyclonal antibodies immunoprecipitate gp300, gp140,gp125, and gp80, whereas mAb 1H8 immunoprecipitate gp300, gp140, andgp80 (FIG. 6). Two cellular proteins (60 and 45 kDa) were alsoassociated with the immune complex preparations using both polyclonaland monoclonal antibodies (FIG. 6, lanes 0 and 2 hr). The presence ofthese 60 and 45 kDa proteins in the immune complex preparation was dueto their binding to protein A Sepharose. This latter was used in orderto recover immune complexes formed with the different antibodies.

[0056] HIV-2 infected cells were pulse-labeled for 1 hr before a chaseof 2 and 4 hr. Extracts from labeled cells (time 0, 2, and 4 hr) and theviral pellet recovered at 4 hr of chase were analyzed byimmunoprecipitation assay using anti-gp300 polyclonal antibodies and mAb1H8 (FIG. 6). With both polyclonal and monoclonal antibodies, gp80 wasnot detectable at the period of pulse-labeling. It became clearlyapparent at 2 hr of chase in infected cells. When the chase wasprolonged to 4 hr, then [³⁵S] methionine labeled gp80 becameundetectable in infected cells. Analysis of viral pellets produced at 4hr, indicated that as the extracellular glycoprotein (gp125), gp80 wasassociated with the virus particles (FIG. 6). These results suggest thatgp80 is a product of the processing of HIV-2 envelope. The fact that wasidentified by monoclonal antibodies specific for the HIV-2 transmembraneglycoprotein indicated that it might be a dimeric form of gp36(confirmed by the results shown in FIGS. 8 and 9). The detection of gp80was not restricted to infected CEM cells, since it was also detectablein HIV-2 infected T4 lymphocytes (data not shown).

[0057] Comparison of the results obtained by Western blot analysis andthe immunoprecipitation assays showed that patient sera, anti-gp300polyclonal antibodies, and mAb 1H8 recognize the denatured forms of gp80and gp125 better than their native forms (FIGS. 1, 2, 3, 4 and 6).Native forms of these mature glycoproteins probably have conformationswhich mask the epitopes identified by the different antibodies.

[0058] VI. Incorporation of Glucosamine and Fucose in gp80

[0059] Extracts from HIV-2 infected cells metabolically labeled with[³H] glucosamine and [³H] fucose were immunoprecipitated using mAb 1H8and anti-gp300 polyclonal antibodies. In accord with the previousresults (FIGS. 3-6), anti-gp300 antibodies immunoprecipitated sugarlabeled gp300, gp140, gp125, and gp80, whereas mAb 1H8immunoprecipitated gp300, gp140, and gp80. All these proteinsincorporated [3H] glucosamine, whereas incorporation of [³H] fucosemainly occured in gp125 and gp80 (FIG. 7a). In these experiments, thelabeling of gp36 with [³H] glucosarmine was observed faintly after aprolonged exposition of the autoradiogram (data not shown; similar toFIG. 1, Rey et al., 1989). The glycoprotein gp80 also can incorporate[³H] mannose as is the case for gp300, gp140, and gp125 (data notshown). Incorporation of these labeled sugars in gp80 and in otherglycoproteins was completely blocked by tunicamycin, an antibiotic whichinhibits N-linked glycosylation of proteins (data not shown).

[0060] Asparagine-linked oligosaccharides (containingN-acetylglucosamine, mannose, and glucose) of glycoproteins undergoextensive processing after their attachment to nascent proteins(Kornfeld and Kornfeld, 1985). Oligosaccharide chains become trimmed inthe endoplasmic reticulum and in the Golgi apparatus before the transferof fucose and sialic acid residues. Therefore, incorporation of fucoseresidues occurs late in the glycosylation pathway. Accordingly, in HIV-2infected cells [³H] fucose becomes incorporated mainly in gp125 andgp80, two proteins which are mature products of the HIV-2 envelopeprecursor (see FIGS. 6 and 7).

[0061] To confirm that gp80 was produced during the processing ofenvelope precursor, HIV-2 infected cells were labeled with [³⁵S]methionine in the absence or presence of the glucosidase inhibitor,castanospermine (Saul et al., 1983). Culture supernatants were thenassayed by immunoprecipitation using an HIV-2 positive patient serumwhich recognizes several viral proteins. In the presence ofcastanospermine, production of gp80 and gp125 were markedly reduced,whereas the production of the major core protein (p26) was notsignificantly affected (FIG. 7b).

[0062] VII. Dissociation of gp80 into gp36

[0063] Preliminary experiments suggested that gp80 could be dissociatedto give gp36. For this reason, experiments were carried out to optimizeconditions under which the dissociation of gp80 might occur, such as,high salt, acidic pH, ionic detergent, EDTA, and EGTA.

[0064] Previously, it was reported that the dimeric form of the envelopeprecursor (gp300) can be dissociated by incubation in slightly acidicbuffer (Rey et al., 1989). Although this latter method also works forgp80, but in a buffer less than pH 6, most of gp80 becomes degraded. Inall experiments, extracts were prepared from infected cells or fromviral pellets by a lysis buffer containing non-ionic detergent, TritonX-100. Under these conditions, gp30 and gp80 are not dissociated evenafter addition of the ionic-detergent SDS.

[0065] The effect of SDS was investigated when it is used instead ofTriton for the preparation of extracts. HIV-infected cells were labeledwith [³⁵S] methionine before preparation of extracts by solubilizationin lysis buffer containing either 1% Triton X-100 or 1% SDS. Theseextracts were then diluted 10 fold in lysis buffer without detergent andimmunoprecipitated using mAb 1H8. The immune-complex preparations fromTriton-extracts showed the presence of [³⁵S] methionine-labeled bandscorresponding to gp300, gp140, gp80 and a faint-band of gp36 (FIG. 8,section C Lane 1). On the other hand, when extracts were prepared withSDS, then gp300 and gp80 were almost undetectable whereas the level ofgp140 and gp36 was increased (FIG. 8, section C lane 2). Thus in thepresence of ionic detergent, the dimeric forms gp300 and gp80 weredissociated giving rise to gp140 and gp36, respectively. Dissociation ofthe purified [³⁵S] methionine labeled gp300 gives only gp140 (Rey etal., 1989). Accordingly, gp36 should arise from the dissociation ofgp80.

[0066] It should be noted that the degradation of proteins occured alsoin the presence of SDS since not all the label in gp300 and gp80 wasrecovered in the dissociated proteins (FIG. 8, section C). The presenceof 200 units/ml aprotinin and 0.2 mM PMSF did not prevent suchdegradation during incubation with SDS. It might be that the dimericforms of proteins have a conformation which can resist proteolysis.Dissociation of gp300 and gp80 might then lead to conformationalmodifications making the proteolytic sites accessible.

[0067] Dissociation experiments were also carried out with the HIV-2pellet. [³⁵S] methionine labeled viral proteins were solubilized inlysis buffer containing 1% Triton or 1% SDS and in RIPA buffercontaining 0.1% SDS and 1% deoxycholate. Extracts in 1% SDS lysis bufferwere also heated at 95° C. All the extracts were then immunoprecipitatedwith mAb 1H8 and analyzed by polyacrylamide gel electrophoresis (FIG. 8,section V). In the immune-complex prepared from Triton-extracts, gp80and gp36 were the major proteins immunoprecipitated by mAb 1H8. On theother hand, gp80 became undetectable whereas the level of gp5 wasslightly increased in SDS-extracts. The degree of degradation inSDS-extracts must have been dramatic since most of the labeldisappeared. A somewhat better result was observed with the RIPA bufferin which most of gp80 was dissociated and about 30% of the label wasrecovered in gp36 (FIG. 8, section V, lane 4).

[0068] In order to show that gp80 is composed of only gp36, dissociationexperiments were carried out using [³⁵S] methionine labeled gp80purified by mAb 1H8 immunoprecipitation and by preparative gelelectrophoresis. Lyophilized gp80 preparations were then suspendeddirectly in an acetate buffer with SDS at OH 6.8, 5.8, and 4.8. At pH4.8, all gp80 was converted to gp36 (FIG. 9).

[0069] VIII. The transmembrane Glycoprotein of SIV-mac Exists in aDimeric Form

[0070] Previously, it was shown that the glycoprotein precursor (gp140)of SIV forms a dimer during its processing (Rey et al., 1989). For thisreason it was important to investigate whether the transmembraneglycoprotein was also detectable as a dimer. SIV and HIV-2 infectedcells were labeled with [³H] glucosamine and extracts prepared by lysisbuffer containing Triton were immunoprecipitated with mAb 1H8. As inFIG. 7, the monoclonal antibody precipitated gp300, gp140, and gp80 fromHIV-2 infected cells. In addition, a very faint band corresponding togp36 was detected (FIG. 10). In SIV infected cells, the monoclonalantibody precipitated three glycosylated proteins: the envelopeprecursor gp140, the dimer precursor gp300, and a 65 kDa protein (gp65)which was probably the counterpart of HIV-2 gp80 (FIG. 10). As gp80,gp65 was found to be associated with SIV virus particles.

[0071] It should be noted that in these experiments, monomeric forms ofthe transmembrane glycoprotein of HIV-2 ROD and SIV-mac were notdetectable. The HIV-2 ROD amino-acid sequence 579-604 corresponds toSIV-mac sequence 595-620 (5,16). Since these two sequences are highlyhomologous, then mAb 1H8 cross-reacts with envelope proteins of bothHIV-2 ROD and SIV-mac.

[0072] By the use of a monoclonal antibody, Veronese et al. (33) haverecently reported that the transmembrane glycoprotein of SIV-mac is a 32kDA protein (gp32). However, in their immunoprecipitation assays, theyreported the presence of unidentified 75 and 300 kDa proteins at highlevels along with the envelope precursor gp140. In analogy with the dataherein, the 75 kDa protein is probably the dimeric form of thetransmembrane glycoprotein gp32 (FIG. 10), whereas the 300 kDa proteinshould be the dimeric form of the envelope precursor previously reported(29).

[0073] This invention thus elucidates the processing of HIV-2 envelopeglycoprotein. The unusual feature of this processing is that theenvelope precursor requires the formation of a homologous dimer in orderto become transported and processed through the Golgi apparatus (Rey etal., 1989). The precursor gp140 becomes dimerized in the roughendoplasmic reticulum and the resulting gp300 dimer intermediaryprecursor is then transported to the Golgi apparatus where it is furtherprocessed. Finally, the dimer is transported to the plasma membrane andcleaved by the cellular protease to yield the mature HIV-2 envelopeglycoproteins: the extracellular glycoproteins (gp125) in monomericforms and the transmembrane glycoproteins (gp36) in dimeric forms (gp80)(FIG. 11).

[0074] Referring to FIG. 11, the envelope polypeptide during itssynthesis becomes glycosylated to give rise to the “hypotheticalglycoprotein precursor” gp150 which becomes rapidly trimmed by the roughendoplasmic reticulum (RER) glucosidases I and II and mannosidase togive rise to gp140. This monomer becomes dimerized and the resultinggp300 “dimer intermediary precursor” is then transported to the Golgiapparatus. Further trimming of gp300 is carried out by the Golgimannosidases before transfer of fucose and sialic acid residues in themedial and trans Golgi. Finally, the dimer is cleaved by the cellularprotease to yield the extracellular envelope glycoprotein (ECG) gp125and the dimeric form of the transmembrane glycoprotein (TMG), gp80.

[0075] In a previous study (Rey et al., 1989), it was suggested that theprocessed gp300 becomes dissociated before transport to the plasmamembrane. This is probably not the case, because now there is evidenceto indicate that the transmembrane proteins become produced ashomodimers. In the previous study, monensin was used as an inhibitor ofthe transport of membrane glycoproteins and secretory proteins from theGolgi apparatus (Johnson and Schlesinger, 1980). In the presence ofmonensin, a 135-kDa protein (gp135), which might have been thedissociated product of gp300, was accumulated in HIV-2 infected cells.This production of gp135 was probably an artifact triggered by monensindue to accumulation of the processed gp300 in the Golgi apparatus.

[0076] The mechanism of dimerization of the envelope glycoproteinprecursor is not yet clear. This is an intrinsic property of thepolypeptide moiety of the envelope precursor. The fact that thetransmembrane glycoproteins exist in dimeric forms (gp80), suggests thatdimerization of gp140 occurs through interactions between thetransmembrane regions of the envelope precursor. Dissociation of thedimeric forms (gp300 an gp80) might occur at slightly acidic pH.Accordingly, dimerization of gp140 might be pH dependent and occurs in acompartment in the rough endoplasmic reticulum which favors the fusionof two gp140 precursor molecules.

[0077] Several observations indicate that formation of gp300 is not anartifact observed in HIV-2 infected CEM cells: (1) Pulse-chaseexperiments in HIV-2 infected CEM cells indicate that gp140 is firstsynthesized before the formation of gp300 which is itself detected fewhours before the production of mature envelope proteins, gp125 and gp80;(2) A similar kinetics for the detection of gp140, gp300, gp125, andgp80 is also observed in T4 lymphocytes infected with HIV-2; (3) Thefate of gp140 is the formation of gp300. This latter is well illustratedby experiments in which transport of the dimer from the endoplasmicreticulum to the Golgi apparatus is blocked by glucosidase inhibitors.In the presence of these inhibitors, all monomer precursors synthesizedby pulse-labeling of infected cells become dimerized during the periodof chase. Dimerization of gp140 is also not a consequence ofexperimental conditions in our studies. Pulse-chase experiments carriedout at reduced temperatures (15-20° C.) to block transport in theendoplasmic reticulum, show that the monomer precursors synthesized bypulse-labeling of infected cells do not form dimers when the chase iscarried out at reduced temperatures. Thus, these results indicate thatformation of gp300 requires transport of gp140 in a compartment with anenvironment favoring the process of dimerization. All these observations(Rey et al., 1989) emphasize that dimerization is a natural step in theprocessing pathway of gp140, i.e., dimerization is not due toaccumulation of unprocessed gp140, nor is it an artifact of theexperimental procedure.

[0078] The molecular weight of the dimeric form of the envelopeprecursor had been estimated by polyacrylamide gel electrophoresis underdenaturing conditions (Rey et al., 1989). In a 5% polyacrylamide gelcontaining 0.1% bisacrylamide instead of 0.2% (wt/vol), this dimericprecursor migrated at a position corresponding to a 280-kDa protein(data not shown). In order to confirm the molecular weight of this dimerunder native conditions, gel filtration experiments were carried outusing an S-300 Sephacryl column and [³⁵S] methionine labeled extractsfrom HIV-2 infected cells prepared by lysis buffer containing Triton.Under these experimental conditions, gp300 eluted as the second peakafter the peak of aggregated proteins. The transmembrane glycoproteindimer eluted as a 75-80 kDA protein after the peak of gp125 and beforethe peak of bovine-serum-albumin (68 kDa) marker (data not shown). Theseobservations indicate that the molecular weight estimations of thenative and denatured dimers gp300 and gp80 are comparable. In vitro, thedissociation of these dimeric forms occured in acidic pH and also in thepresence of the ionic-detergent SDS. When extracts were prepared in thelysis buffer containing Triton, then the dimeric forms gp300 and gp80resisted dissociation by SDS. The non-ionic detergent Triton, therefore,conserved the native forms of the envelope dimers, gp300 and gp80.

[0079] Dimeric forms of the envelope precursor and the transmembraneglycoprotein were also observed in cells infected with SIV but not withHIV-1. In the case of HIV-1, it has been reported that a smallproportion of the transmembrane glycoprotein in the HIV-1 particlesmight exist as a dimer linked by disulfide bonds (Bharat Parekh andRoger Walker, personal communication). The transmembrane dimers of HIV-2(gp80) and SIV (gp65), however, resist dissociation by reducing agents.Accordingly, dimerization of the envelope glycoprotein precursor can beconsidered a specific property of HIV-2 and SIV envelope geneexpression. This property could be used for characterization of new HIVisolates, as a convenient marker to describe their relationship to HIV-1or HIV-2. Dimerization of the envelope precursor might be required forthe processing of the envelope precursor to yield the mature envelopeglycoproteins. The dimeric form of the transmembrane glycoprotein mightbe essential for optimal structure of the virion and thus its capacityto fuse with the cellular membrane and be infectious. In vitrodissociation of the transmembrane dimer leads to a dramatic degradation.It might be, therefore, that the dimeric forms of this glycoprotein havea conformation which resists proteolysis.

[0080] Previously, the formation of oligomeric complexes of some viralstructural glycoproteins has been reported. For example, thehemagglutinin (HA) of influenza virus exists as a trimer which could bestabilized by cross-linking agents (15, 36, 37), the G glycoprotein ofthe vesicular stomatitis virus (VSV) also forms a trimer (11, 24). Morerecently, the envelope glycoprotein precursor of the Rous sarcoma viruswas reported to form a trimer held by a disulfide linkage (12). In allthese studies, formation of oligomeric complexes has been associatedwith their intracellular transport from the endoplasmic reticulum. Inthe case of the RSV the trimeric structure of the transmembraneglycoprotein seems to be the functional form found in virions.Therefore, the results observed in the RSV are analogous with thosepresented here, i.e., oligomerization of the HIV-2 and SIV envelopeglycoprotein precursor and the transmembrane glycoprotein. In contrastto the RSV however, the dimeric forms of the HIV-2 and SIV are stableand are not dissociated by reducing agents. Nevertheless, all theseobservations enforce the suggestion that efficient processing of someglycoproteins requires the formation of oligomeric structures, and insome cases oligomeric forms of the mature glycoprotein might beessential for infectivity. Accordingly, antiviral agents designed toblock the formation of oligomeric precursors or cause dissociation ofoligomeric complexes of the mature proteins, might be employed toprevent virus replication and its spreading.

[0081] Following is a more detailed description of the experimentalProcedures used in this invention.

[0082] Materials

[0083] L-[³⁵S] Methionine (specific activity>1000 μCi/mmol, L-[6-³H]Fucose (specific activity: 45-70 μCi/mmol), D-[6-³H] Glucosamine(specific activity: 20-40 μCi/mmol) were purchased from Amersham(Amersham, UK). Castanospermine and tunicamycin were obtained fromBoehringer-Mannheim (Manneheim, West Germany). Poly(A).poly(U) was thegenerous gift of M. Michelson, Institut Curie, Paris, and is preparedaccording to Hovanessian et al. (J. Interferon Res. 1982, 2:209-216).

[0084] Virus and Cells

[0085] HIV-1_(BRU) isolate of the human immunodeficiency virus type 1(Montagnier et al., 1984), HIV-2_(ROD) isolate of the humanimmunodeficiency virus type 2 (Clavel et al., 1986), and Simianimmunodeficiency virus, SIVmac₁₄₂ (Daniel et al., 1985) were used inthis study.

[0086] The different cell lines and human lymphocytes were cultured insuspension medium RPMI-1640 (GIBCO-BRL, Cergy-Pontoise France)containing 10% (v/v) fetal calf serum; 2 μg/ml Polybrene (Sigma) wasadded for HIV infected cell cultures. CEM clone 13 cells are derivedfrom the human lymphoid cell line CEM (ATCC-CCL119) and express the T4antigen to a high level. Five days after infection with HIV-1_(BRU) orHIV-2_(ROD) isolates, about 80-90% of the cells produce viral particlesand can be identified by a cytopathic effect corresponding tovacuolisation of cells and appearance of small syncitia. The HUT-78 cellline is another human T4 positive lymphoid cell line (Gazdar et al.,1980) that is highly permissive for the replication of SIVmac₁₄₂ (Danielet al., 1985). Peripheral blood lymphocytes from healthy blood donorswere stimulated for three days with 0.2% (w/v) phytohemagglutininfraction P (Difco, Detroit, USA) in RPMI-1640 medium supplemented with10% fetal calf serum. Cells were then cultured in RPMI-1640 mediumcontaining 10% (v/v) T cell growth factor (TCGF, Biotest). Afterinfection with HIV-2, lymphocytes were cultured in presence of 10% (v/v)TCGF and 2 μg/ml Polybrene.

[0087] Metabolic Labeling of Cells

[0088] For metabolic labeling of proteins, infected cells were incubatedfor 16 hours at 37° C. in MEM culture without L-methionine and serum,but supplemented with 200 μCi/ml [³⁵S] methionine. For metaboliclabeling of glycoproteins, infected cells were incubated for 16 hours at37° C. in MEM culture medium lacking serene and glucose but supplementedwith 200 μCi/ml ³H-Fucose or 200 μCi/ml ³H-glucosamine.

[0089] Cells and Viral Extracts

[0090] Cell pellets corresponding to 10⁷ cells were resuspended in 100μl of buffer: 10 mM Tris-HCl pH 7.6, 150 mM NaCl, 1 mM EDTA, 0.2 mMPMSF, 100 units/ml aprotinin (Iniprol, Choay) before addition of 100 μlof the same buffer containing 2% (v/v) Triton X-100. Cell extracts werecentrifuged at 12,000 a for 10 minutes, and the supernatant was storedat −80° C. until used. For viral extract preparations, 100 μl of10×lysis buffer (100 mM Tris-HCl pH 7.6, 1.5M NaCl, 10 mM EDTA, 10%(v/v) Triton X-100, 100 units/ml aprotinin) were added per ml ofclarified supernatant from infected CEM cells and processed as above.For the preparation of extracts from virus pellets, culture medium frominfected cells was first centrifuged at 12,000 g for 10 minutes beforehigh speed centrifugation at 100,000 g for 30 to 60 min. Virus pellets(material from 10⁷ cells) were then solubilized in 200 μl of lysisbuffer.

[0091] Preparation of an Immunoadsorbant with Antibodies from an HIV-2Seropositive Patient

[0092] Immunoglobulins from the serum of an HIV-2 seropositive patientwere precipitated with 50% (NH₄)₂SO₄, dissolved in 20 mM sodiumphosphate (pH 8.0) and further purified on a DEAE cellulose column(DE52, Whatman) by elution with 20 mM sodium phosphate (pH 8.0).Immunoglobulins purified in this manner were judged to be 90% pure. Theantibodies were subsequently coupled to CNBr-activated Sepharose CL 4Baccording to a technique described (Berg, 1977). Two milligrams of IgGwere coupled per ml of Sepharose CL 4B. This immunoadsorbant is referredto as HIV-2 serum-Sepharose.

[0093] Preparation of HIV-2 Proteins on an immunoaffinity Column

[0094] Cell extracts from HIV-2 producing CEM cells were first dilutedin two volumes of binding buffer (20 mM Tris-HCl pH 7.6, 50 mM KCl, 150mM NaCl, 1 mM EDTA, 1% (v/v) glycerol, 7 mM 6-mercaptoethanol, 0.2 mMPMSF, 100 units/ml aprotinin) before incubation with one volume of HIV-2serum-Sepharose. Supernatants from HIV-2 producing cells were processedas cell extracts except that only one tenth of binding bufferconcentrate 10× was added per volume of supernatant. The binding wascarried out overnight, then the column was washed batchwise in bindingbuffer. Proteins bound to the column were eluted by boiling inelectrophoresis sample buffer (125 mM Tris-HCl pH 6.8, 1% (w/v) SDS, 2Murea, 20% glycerol, 0.5% β-mercaptoethanol). Eluted proteins wereresolved by electrophoresis on 7.5% polyacrylamide-SDS gels containing6M urea and 0.1% bisacrylamide instead of 0.2% (w/v).

[0095] Preparative Electrophoresis

[0096] HIV-2 glycoproteins (gp300 or gp80) eluted from the affinitycolumn were resolved by polyacrylamide gel electrophoresis as previouslydescribed, and the regions of the gel containing the viral glycoproteinswere cut out by reference to the position of prestained molecular weightprotein markers (BRL). Glycoprtein gp300 was eluted by incubation for 16hours at 4° C. in elution buffer (0.1M NaHCO₃, 0.5 mM EDTA, 0.05% (w/v)SDS, 0.2 mM PMSF). The glycoprotein fractions thus obtained werelyophilized and kept refrigerated until used. Glycoprotein gp80 waselectroeluted in buffer containing 4 mM Tris-HCl pH 7.6, 2 mM sodiumacetate, and 2 mM EDTA.

[0097] Preparation of Murine Polyclonal Antibodies, Anti-gp300

[0098] HIV-2 envelope glycoprotein gp300 was purified from extracts ofinfected CEM cells (3×10⁸ cells) by immunoaffinity chromatography on theHIV-2 serum-Sepharose and followed by preparative gel electrophoresis(Rey et al., 1989). The purified preparation of gp300 was dissolved in10 ml of 150 mM NaCl containing 0.5M urea and 1 mg/ml of mouse serumproteins and dialyzed for 24 hours against the solution containing 150mM NaCl and 0.5M urea. The dializate was then centrifuged and 2 mlaliquots were stored at −80° C.

[0099] Five mice (8 weeks old) were injected intraperitoneally, fivetimes at 12 days interval with 350 μl of the gp300 preparation (about0.1 μg of gp300). Poly(A).poly(U) (200 μg; 1 mg/ml in 150 mM NaCl) wasused as an adjuvant which was administered intravenously during eachimmunization (Hovanessian et al., 1988). Five days before the lastinjection, mice were injected intraperitoneally with a suspension of 10⁶sarcoma 180/TG cells to prepare hyperimmune ascitic fluid (Hovanessianet al., 1988 A week following the booster, mice were sacrificed and theascitic fluids were collected. Ascitic cells were removed bycentrifugation (200 g, 5 min) and the peritoneal fluid was collected.

[0100] Production and Characterization of Monoclonal, mAb 1H8

[0101] HIV-2 ROD virions were cultivated in CEM cells and purified fromconcentrated culture supernatants by banding in sucrose gradients.Purified virus was disrupted in 0.5% Triton X-100, 150 mM NaCl, 50 mMTris, pH 8.0, 1% aprotinin (Sigma) and clarified byultra-centrifugation. The viral extract was then passed over aLentil-Lectin Sepharose 4B affinity column (Pharmacia), the columnwashed, and the bound glycoproteins eluted with 0.5 M methyla-D-mannopyranoside (Sigma), and dialyzed overnight againstphosphate-buffered saline. BALB-C mice were immununizedintraperitoneally with 0.3 mis of purified glycoproteins (2-54 g)Reattached to Lentil-Lectin Sepharose 4B (50-100 μl). The mice wereboosted every 4-6 weeks for 24 weeks with the same immunogen andmonitored for HIV-2 glycoprotein antibodies qualitatively by RIPA of[35S] methionine labeled HIV-2 virion extracts and quantitatively on theGenetic Systems HIV-2 disrupted virion EIA. Three days after the lastinjection, spleen cells were fused with NSI myeloma cells according tothe method of Kohler and Milstein 22). 96-well fusion plates werescreened by hybridomas secreting anti-HIV-2 antibodies using the GeneticSystems HIV-2 disrupted virion EIA. Methods for the propagation andstabilization of cloned hybridomas and for ascites production have beenpreviously described (16). Hybridoma culture supernatants were screenedby RIPA and Western blot analysis. Monoclonal antibody (mAb) 1H8, whichreacted with the transmembrane glycoprotein, was further mapped toamino-acid squence 579-604 within the HIV-2 transmembrane glycoproteinusing a synthetic peptide based EIA (32). The HIV-2 amino-acid sequence579-604 is highly conserved among all HIV-2 and SIV isolates thus farsequenced accordingly, monoclonal antibody 1H8 cross-reacts with HIV-2and SIV isolates thus far tested. The synthetic peptide p39′ wassynthesized according to the amino-acid sequence 579-604 deduced fromthe nucleotide sequence of the HIV-2 ROD envelope. The amino-acidsequence of peptide p39′ is the following VTAIEKYLQDQARLNSWGCAFRQVCH.

[0102] Radio-Immunoprecipitation Assay (RIPA)

[0103] Cell or viral extracts (20 μl) (material corresponding to 1×10⁶infected cells) were first diluted in two volumes of RIPA buffer [(10 mMTris-HCl pH 7.6, 150 mM NaCl, 1M EDTA, 1% Triton X-100 (v/v), 0.2%sodium deoxycholate (wt/v), 0.1% SDS (wt/v), 7 mM 2β-mercaptoethanol,0.2 mM PMSF, 100 units/ml of aprotinin (Iniprol, Choay)]. Dilutedextracts were then incubated (45 min, 4° C.) with polyclonal ormonoclonal antibodies (2-5 μl). Protein A-Sepharose was then added andthe samples were further incubated for 3 hr at 4° C. These samples werewashed in the RIPA buffer. Proteins recovered by immunoprecipitationwere eluted by heating (95° C., 5 min) in the electrophoresis samplebuffer [125 mM Tris-HCl, pH 6.8, 1% SDS (wt/v), 20% glycerol (v/v), 1% 2β-mercaptoethanol]. Eluted proteins were resolved by electrophoresis in7.5-12.5% polyacrylamide SDS gels containing 0.1% bisacrylamide insteadof 0.2% (wt/v).

[0104] Electrophoretic Transfer Immunoblot Analysis: Western Blot

[0105] Proteins were subjected to analysis by polyacrylamide gelelectrophoresis before being electrophoretically transferred to 0.45 μmnitrocellulose sheets (Schleicher and Schull, Dassel, FRG) in electrodebuffer (20 mM Tris base, 150 mM glycine, 20% methanol, v/v) as described(Burnette 1981). The electrophoretic blots were saturated with 5% (w/v)non-fat dry milk in PBS (Johnson et al., 1984). They were then incubatedin a sealed bag (overnight 4° C.) either with HIV-1 or HIV-2 positivesera (at 1:100 dilution) or with mouse polyclonal or monoclonalantibodies (at 1:200 dilution) in PBS containing 10% FCS. The sheetswere subsequently washed in PBS, PBS containing 5% Nonidet P-40 and thenresaturated in PBS containing non-fat milk (5%). The washed sheets werethen incubated (2 hr, room temperature) in a sealed bag either with apreparation of ¹²⁵I-labeled protein A (Amersham, >30 mCi/mg) to revealthe human polyclonal antibodies in the HIV-1 or HIV-2 sera or with apreparation of ¹²⁵I-labeled goat anti-mouse immunoglobulins (Amersham;2-10 μCi/μg). The sheets were removed from the bags and washed again,dried and autoradiographed (Kodak RP Royal, X-Ray films) for 24-48 hr.

[0106] It will be understood that the present invention is intended toencompass the previously described proteins and glycoproteins inpurified form, whether or not fully glycosylated, and whether obtainedusing the techniques described herein or other methods. In a preferredembodiment of this invention, the retrovirus and polypeptides aresubstantially free of human tissue and human tissue components, nucleicacids, extraneous proteins and lipids, and adventitious microorganisms,such as bacteria and viruses. It will also be understood that theinvention encompasses equivalent proteins and glycoproteins havingsubstantially the same biological and immunogenic properties. Thus, thisinvention is intended to cover serotypic variants of the proteins andglycoproteins of the invention.

[0107] The proteins and glycoproteins of this invention can be obtainedby culturing HIV-2 in susceptible mammalian cells of lymphocyticlineage, such as T-lymphocytes or pre-T-lymphocytes of human origin ornon-human primate origin (e.g. chimpanzee, African green monkey, ormacaques.) A number of different lymphocytes expressing the CD4phenotypic marker can be employed. Examples of suitable target cells forHIV-2 infection are mononuclear cells prepared from peripheral blood,bone marrow, and other tissues from patients and donors. Alternatively,established cell lines can be employed. For example, HIV-2 can bepropagated on blood-donor lymphocyte cultures, followed by propagationon continuous cell strains of leukemic origin, such as HUT 78. HUT 78 isa well characterized mature human T cell line, which has been depositedat Collection Nationale Des Cultures De Micro-organismes (CNCM) at thePasteur Institut in Paris, France on Feb. 6, 1986, under culturecollection deposit accession number CNCM 1-519. Another suitable targetor HIV-2 infection and production of the proteins and glycoproteins ofthe invention is the T-cell line derived from an adult with lymphoidleukemia and termed HT. HT cells continuously produce virus afterparental cells are repeatedly exposed to concentrated cell culturefluids harvested from short-term culture T-cells grown in TCGF thatoriginated from patients with LAS or AIDS, in addition, there areseveral other T or pre-T human cell lines such as CEM and MOLT 3 thatcan be infected and continue to produce HIV-2. Furthermore,B-lymphoblastic cell lines can also be productively infected by HIV.Montagnier et al, Science, 225:63-66(1984).

[0108] The proteins and glycoproteins of the invention can be producedin the target cells using the culture conditions previously described,as well as other standard techniques. For instance, infected humanlymphocytes can be stimulated for three days by phytohemaglutinin (PHA).The lymphocytes can be cultured in RPMI-1640 medium to which has beenadded 10% fetal calf serum, 10⁻⁵M beta mercaptoethanol, interleukin 2,and human alpha anti-interferon serum. Barre-Sinoussi et al, Science,220:868-871 (1983). In addition, techniques for the-propagation of HIV-2in RUT 78 and CEM cell lines are described in copending U.S. applicationSer. No. 835,228, filed Mar. 3, 1986, the entire disclosure of which isrelied upon and incorporated by reference herein.

[0109] The production of virus in the cell cultures can be monitoredusing several different techniques. Supernatant fluids in the cellcultures can be monitored for viral reverse transcriptase activity.Electron microscopic observation of fixed and sectioned cells can alsobe used to detect virus. In addition, virus can be detected bytransmitting the virus to fresh normal human T-lymphocytes (e.g.,umbilical cord blood, adult peripheral blood, or bone marrow leukocytes)or to established T-cell lines. Testing for antigen expression byindirect immunofluorescence or Western Blot procedures using serum fromseropositive donors can also be employed. In addition, nucleic acidprobes can be utilized to detect viral production.

[0110] After a sufficient period of time for viral multiplication totake place, infected cells can be separated from the culture medium anddisrupted to expose intracellular proteins using conventionaltechniques. For example, physical shearing, homogenization, sonication,detergent solublization, or freeze-thawing can be employed. The viralproteins released by these cells can be separated from the othercellular components and purified using standard biochemical procedures.For example, virus can be recovered by ultra centrifugation, and theviral proteins can then be solubilized by detergent and then purified bygel filtration, ion-exchange chromatography, affinity chromatography,dialysis, or by the use of monoclonal antibodies or by combinations ofthese procedures. A thorough purification of the antigens of theinvention can be performed by immunoreaction with the sera of patientsknown to possess antibodies effective against the antigens, withconcentrated antibody preparations such as polyclonal antibodies, orwith monoclonal antibodies directed against the antigens of theinvention.

[0111] The proteins and the glycoproteins of the present invention canbe used as antigens to identify antibodies to HIV-2 and SIV in materialsand to determine the concentration of the antibodies in those materials.Thus, the antigens can be used for qualitative or quantitativedetermination of the retrovirus in a material. Such materials of courseinclude human tissue and human cells, as well as biological fluids, suchas human body fluids, including human sera. When used as a reagent in animmunoassay for determining the presence or concentration of theantibodies to HIV-2, the antigens of the present invention provide anassay that is convenient, rapid, sensitive, and specific.

[0112] More particularly, the antigens of the invention can be employedfor the detection of HIV-2 by means of immunoassays that are well knownfor use in detecting or quantifying humoral components in fluids. Thus,antigen-antibody interactions can be directly observed or determined bysecondary reactions, such as precipitation or agglutination. Inaddition, immunoelectrophoresis techniques can also be employed. Forexample, the classic combination of electrophoresis in agar followed byreaction with anti-serum can be utilized, as well as two-dimensionalelectrophoresis, rocket electrophoresis, and immunelabeling ofpolyacrylamide gel patterns (Western Blot or immunoblot.) Otherimmunoassays in which the antigens of the present invention can beemployed include, but are not limited to, radioimmunoassay, competitiveimmunoprecipitation assay, enzyme immunoassay, and immunofluorescenceassay. It will be understood that tubidimetric, calorimetric, andnephelometric techniques can be employed. An immunoassay based onWestern Blot technique is preferred.

[0113] Immunoassays can be carried out by immobilizing one of theimmunoreagents, either an antigen of the invention or an antibody of theinvention to the antigen, on a carrier surface while retainingimmunoreactivity of the reagent. The reciprocal immunoreagent can beunlabeled or labeled in such a manner that immunoreactivity is alsoretained. These techniques are especially suitable for use in enzymeimmunoassays, such as enzyme linked immunosorbent assay (ELISA) andcompetitive inhibition enzyme immunoassay (CIEIA).

[0114] When either the antigen of the invention or antibody to theantigen is attached to a solid support, the support is usually a glassor plastic material. Plastic materials molded in the form of plates,tubes, beads, or disks are preferred. Examples of suitable plasticmaterials are polystyrene and polyvinyl chloride. If the immunoreagentdoes not readily bind to the solid support, a carrier material can beinterposed between the reagent and the support. Examples of suitablecarrier materials are proteins, such as bovine serum albumin, orchemical reagents, such as gluteraldehyde or urea. Coating of the solidphase can be carried out using conventional techniques.

[0115] Depending on the use to be made of the proteins and glycoproteinsof the invention, it may be desirable to label them. Examples ofsuitable labels are radioactive labels, enzymatic labels, fluorescentlabels, chemiluminescent labels, and chromophores. The methods forlabeling proteins and glycoproteins of the invention do not differ inessence from chose widely used for labeling immunoglobulin. The need tolabel may be avoided by using labeled antibody to the antigen of theinvention or anti-immunoglobulin to the antibodies to the antigen as anindirect marker.

[0116] Once the proteins and glycoproteins of the invention have beenobtained, they can be used to produce polyclonal and monoclonalantibodies reactive therewith. Thus, a protein or glycoprotein of theinvention can be used to immunize an animal host by techniques known inthe art. Such techniques usually involve inoculation, but they mayinvolve other modes of administration. A sufficient amount of theprotein or the glycoprotein is administered to create an immunogenicresponse in the animal host. Any host that produces antibodies to theantigen of the invention can be used. Once the animal has been immunizedand sufficient time has passed for it to begin producing antibodies tothe antigen, polyclonal antibodies can be recovered. The general methodcomprises removing blood from the animal and separating the serum fromthe blood. The serum, which contains antibodies to the antigen, can beused as an antiserum to the antigen. Alternatively, the antibodies canbe recovered from the serum. Affinity purification is a preferredtechnique for recovering purified polyclonal antibodies to the antigen,from the serum.

[0117] Monoclonal antibodies to the antigens of the invention can alsobe prepared. One method for producing monoclonal antibodies reactivewith the antigens comprises the steps of immunizing a host with theantigen; recovering antibody-producing cells from the spleen of thehost; fusing the antibody-producing cells with myeloma cells deficientin the enzyme hypoxanthine-guanine phosphoribosyl transferase to formhybridomas; selecting at least one of the hybridomas by growth in amedium comprising hypoxanthine, aminopterin, and thymidine; identifyingat least one of the hybridomas that produces an antibody to the antigen;culturing the identified hybridoma to produce antibody in a recoverablequantity; and recovering the antibodies produced by the culturedhybridoma.

[0118] These polyclonal or monoclonal antibodies can be used in avariety of applications. Among these is the neutralization ofcorresopnding proteins. They can also be used to detect viral antigensin biological preparations or in purifying corresponding proteins,glycoproteins, or mixtures thereof, for example when used in affinitychromatographic columns.

[0119] The invention provides immunogenic proteins and glycoproteins,and more particularly, protective polypeptides use in the preparation ofvaccine compositions against HIV-2. These polypeptides can thus beemployed as viral vaccines by administering the polypeptides to a mammalsusceptible to HIV-2 infection. Conventional modes of administration canbe employed. For example, administration can be carried out by oral,respiratory, or parenteral routes. Intradermal, subcutaneous, andintramuscular routes of adminstration are preferred when the vaccine isadministered parenterally.

[0120] The ability of the proteins, glycoproteins, and vaccines of theinvention to induce protective levels of neutralizing antibody in a hostcan be enhanced by emulsification with an adjuvant, incorporation in aliposome, coupling to a suitable carrier, or by combinations of thesetechniques. For example, the proteins and glycoproteins of the inventioncan be administered with a conventional adjuvant, such as aluminumphosphate and aluminum hydroxide gel, in an amount sufficient topotentiate humoral or cell-mediated immune response in the host.Similarly, the polypeptides can be bound to lipid membranes orincorporated in lipid membranes to form liposomes. The use ofnonpyrogenic lipids free of nucleic acids and other extraneous mattercan be employed for this purpose.

[0121] The immunization schedule will depend upon several factors, suchas the susceptibility of the host to infection and the age of the host.A single dose of the vaccine of the invention can be administered to thehost or a primary course of immunization can be followed in whichseveral doses at intervals of time are administered. Subsequent dosesused as boosters can be administered as needed following the primarycourse.

[0122] The proteins and vaccines of the invention can be administered tothe host in an amount sufficient to prevent or inhibit HIV-2 infectionor replication in vivo. In any event, the amount administered should beat least sufficient to protect the host against substantialimmunosuppression, even though HIV infection may not be entirelyprevented. An immunogenic response can be obtained by administering theproteins or glycoproteins of the invention to the host in an amount ofabout 10 to about 500 micrograms antigen per kilogram of body weight,preferably about 50 to about 100 micrograms antigen per kilogram of bodyweight. The proteins and vaccines of the invention can be administeredtogether with a physiologically acceptable carrier. For example, adiluent, such as water or a saline solution, can be employed.

[0123] Reference is made herein to proteins of the invention. Theproteins of the invention include, for example, polypeptides that arenot glycosylated. The proteins can be prepared using conventionaltechniques. For instance, glycosylation of the proteins in vivo can beblocked by tunicamycin, an antibiotic which inhibits N-linkedglycosylation of proteins (Schwartz et al., 1976; Kornfeld and-Kornfeld,1985). Alternatively, glycoproteins of the invention can bedeglycosylated by β-N-acetylglucosaminase H (endo H), which cleaves highmannose-type oligosaccharide chains (Tarentino et al., J. Biol. Chem.,249: 818-824, 1974).

[0124] In summary, proteins and glycoproteins, which are precursors ofHIV-2 and SIV envelope glycoprotein and the dimeric form of thetransmembrane glycoprotein, have now been identifed. In addition toproviding useful tools for detection of antibodies to the retrovirus inhumans and for raising neutralizing antibodies to HIV-2 in vitro and invivo, this invention adds to the base of knowledge relating toimmunodeficiency active proteins and glycoproteins of the AIDS viruses.

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What is claimed is:
 1. HIV-2 virus containing a structural glycoprotein of 80 kDa, wherein said virus is in a purified Form.
 2. A glycoprotein of human immunodeficiency virus type 2 (HIV-2) wherein (A) said glycoprotein is dimeric form of tine transmembrane glycoprotein of HIV-2; (B) said glycoprotein has an apparent molecular weight of about 80 kDa (gp80); and (C) said glycoprotein is in a purified form.
 3. A glycoprotein of simian immunodeficiency virus (SIV) or of a simian retroviral variant of SIV, wherein (A) said glycoprotein is dimeric form of the transmembrane glycoprotein of SIV; (B) said glycoprotein has an apparent molecular weight of about 65 kDa (gp65SIV); and (C) said glycoprotein is in a purified form.
 4. A non-glycosylated dimeric transmembrane envelope protein of HIV-2 in a purified form.
 5. A labeled antigen-comprising a glycoprotein of human immunodeficiency virus type 2 (HIV-2), wherein (A) said glycoprotein is a dimeric form of the transmembrane protein of HIV-2; (B) said glycoprotein has an apparent molecular weight of about 80 kDa (gp80); and (C) said antigen is labeled with an immunoassay label selected from the group consisting of radioactive, enzymatic, fluorescent, chemiluminescent labels, and chromophores.
 6. The labeled antigen of claim 5, which is non-glycosylated.
 7. The labeled antigen of claim 5, wherein the antigen is in a purified form.
 8. A labeled antigen comprising a glycoprotein of simian immunodeficiency virus (SIV) or of a simian retroviral variant of SIV, wherein (A) said glycoprotein is dimeric form of the transmembrane protein of SIV; (B) said glycoprotein has an apparent molecular weight of about 65 kDa (gp65_(SIV)); and (C) said antigen is labeled with an immunoassay label selected from the group consisting of radioactive, enzymatic, fluorescent, chemiluminescent labels, and chromophores.
 9. The labeled antigen of claim 8, wherein the antigen in a purified form.
 10. An immunological complex between the glycoprotein of claim 2 and an antibody recognizing said glycoprotein.
 11. An immunological complex between the glycoprotein of claim 3 and an antibody recognizing said glycoprotein.
 12. An immunological complex between the antigen of claim 4 and an antibody recognizing said antigen.
 13. An immunological complex between the antigen or claim 5 and an antibody recognizing said antigen.
 14. An immunological complex as claimed in claim 10, wherein the antibody is labeled with an immunoassay label selected from the group consisting of radioactive, enzymatic, fluorescent, chemiluminescent labels, and chromophores.
 15. An immunological complex as claimed in claim 11, wherein the antibody is labeled with an immunoassay label selected from the group consisting of radioactive, enzymatic, fluorescent, chemiluminescent labels, and chromophores.
 16. A method for detecting infection of cells by human immunodeficiency virus type-2 (HIV-2), wherein the method comprises: (A) providing a composition comprising cells suspected of being infected with HIV-2; (B) disrupting cells in the composition to expose intracellular proteins; and (C) assaying the exposed intracellular proteins for the presence of gp80 glycoprotein of HIV-2.
 17. The method of claim 16, wherein said assaying of exposed intracellular proteins is carried out by electrophoresis of said proteins.
 18. The method of claim 16, wherein said assaying of exposed intracellular proteins is carried out by polyacrylamide gel electrophoresis of said proteins.
 19. The method of claim 16, wherein said assaying of exposed intracellular proteins is carried out by immunoassay with antibodies that are immunologically reactive with gp80 glycoprotein of HIII-2.
 20. The method of claim 19, wherein said antibodies are labeled with an immunoassay label.
 21. A method of detecting antigens of human immunodeficiency virus type-2 (HIV-2), wherein the method comprises: (A) providing a composition suspected of containing antigens of HIV-2; and (B) assaying said composition for the presence of gp80 glycoprotein of HIV-2.
 22. The method of claim 21, wherein said assaying of said composition is carried out by electrophoresis of said proteins.
 23. The method of claim 21, wherein said assaying of said composition is carried out by polyacrylamide gel electrophoresis of said proteins.
 24. The method of claim 21, wherein said assaying of said composition is carried out by immunoassay with antibodies that are-immunologically reactive with gp80 glycoprotein of HIV-2.
 25. The method of claim 24, wherein said antibodies are labeled with an immunoassay label.
 26. A method of distinguishing HIV-2 infection from HIV-1 infection in cells suspected of being infected therewith, wherein the method comprises: (A) providing in extract containing intracellular proteins of said cells; and (B) assaying said extract for the presence of gp80 glycoprotein of HIV-2.
 27. The method of claim 26, wherein said assaying of said extract is carried out by electrophoresis of said proteins.
 28. The method of claim 26, wherein said assaying of said extract is carried out by polyacrylamide gel electrophoresis of said proteins.
 29. The method of claim 26, wherein said assaying of said extract is carried out by immunoassay with antibodies that are immunologically reactive with gp80 glycoprotein of HIV-2.
 30. The method of claim 29, wherein said antibodies are labeled with an immunoassay label.
 31. A method of making gp80 glycoprotein of human immunodeficiency virus type-2 (HIV-2), wherein the method comprises: (A) providing a composition containing cells in which HIV-2 is capable of replicating; (B) infecting said cells with HIV-2; (C) culturing said cells under conditions to cause HIV-2 to proliferate; (D) disrupting said cells to expose intracellular proteins; and (E) recovering gp80 glycoprotein from the resulting exposed intracellular proteins.
 32. A method of making gp80 glycoprotein of HIV-2, wherein the method comprises detergent solubilization of HIV-2 virions.
 33. An in vitro diagnostic method for the detection of the presence or absence of antibodies which bind to an antigen comprising the glycoprotein of claim 2, which method comprises: contacting said antigen with a biological fluid for a time and under conditions sufficient for the retroviral antigen and antibodies in the biological fluid to form an antigen-antibody complex; and detecting the formation of the complex.
 34. The method of claim 33, wherein the detecting step further comprises measuring the formation of said antigen-antibody complex.
 35. An in vitro diagnostic method for the detection of the presence or absence of antibodies which are capable of binding to an antigen comprising the glycoprotein of claim 3, which method comprises: contacting said antigen with a biological fluid for a time and under conditions sufficient for the retroviral antigen and antibodies-in the biological fluid to form an antigen-antibody complex; and detecting the formation of the complex.
 36. The method of claim 35, wherein the detecting step further comprises measuring the formation of said antigen-antibody complex.
 37. A method for detection of antibodies which specifically bind to antigenic sites of an antigen indicative of the presence of the glycoprotein of claim 3 in a sample of human body fluid, which comprises: contacting said antigen with antibodies from human body fluid for a time and under conditions sufficient to permit formation of an antigen-antibody complex between said antigen and said antibodies; and measuring the formation of said antigen-antibody complex by immunoassay based on Western Blot technique or ELISA (enzyme linked immunosorbent assay) or indirect immunofluorescent assay.
 38. A diagnostic kit for the detection of the presence or absence of antibodies which are capable of binding to a glycoprotein as claimed in claim 2, which kit comprises: antigens comprising said glycoprotein; and means for detecting the formation of immune complex between said antigens and said antibodies; wherein the antigens and the means are present in an amount sufficient to perform said detection.
 39. The diagnostic kit of claim 38, wherein the means for detecting the formation of the immune complex are immunological assay means selected from the group consisting of radioimmunoassay, immunoenzymatic, and immunofluorescent assay means.
 40. A diagnostic kit for the detection of the presence or absence of antibodies which are capable of binding to a glycoprotein as claimed in claim 3, which kit comprises: antigens comprising said glycoprotein; and means for detecting the formation of immune complex between said antigens and said antibodies; wherein the antigens and the means are present in an amount sufficient to perform said detection.
 41. The diagnostic kit of claim 40, wherein the means for detecting the formation of the immune complex are immunological assay means selected from the group consisting of radioimmunoasssay, immunoenzymatic, and immunofluorescent assay means.
 42. A method for the preparation of dimeric form of the transmembrane glycoprotein of human immunodeficiency virus type 2 (HIV-2), wherein the method comprises: (A) providing an extracellular composition containing gp80 glycoprotein of HIV-2; and (B) dissociating the gp80 glycoprotein into gp36 glycoprotein of HIV-2.
 43. The method of claim 42, wherein said gp80 is dissociated in a buffer less than about pH
 6. 44. The method of claim 43, wherein the pH is about 4.8.
 45. The method of claim 42, wherein said gp80 is dissociated in an ionic detergent.
 46. An immunogenic composition comprising a pharmaceutically effective amount of a glycoprotein of claim 2 in association with a pharmaceutically acceptable carrier therefor.
 47. An immunogenic composition comprising glycoprotein of claim 2 or a non-glycosylated protein thereof capable of eliciting antibody production.
 48. A vaccine composition comprising a neutralizing amount of dimeric transmembrane envelope glycoprotein or unglycosylated form thereof of HIV-2 and a pharmaceutically acceptable carrier therefor.
 49. A diagnostic kit for the detection of the presence or absence of antibodies which bind to antigens indicative of human immunodeficiency virus type 2 (HIV-2), wherein said kit comprises glycosylated or non-glycosylated gp80 or mixtures thereof and means for detecting the formation of immune complexes between said antigens and said antibodies, wherein the antigens and means are present in an amount sufficient to perform said detection. 